Selective cb2 receptor agonists for use in the prevention or treatment of alcoholic liver disease

Title: Selective cb2 receptor agonists for use in the prevention or treatment of alcoholic liver disease.Abstract: The present invention relates to selective CB2 agonists for use in the prevention or treatment of alcoholic liver disease and particularly alcoholic hepatic steatosis and liver inflammation, and pharmaceutical composition thereof. ...

BACKGROUND OF THE INVENTION

Alcoholic liver disease is one of the major medical complications of alcoholic abuse. Currently, alcohol accounts for the majority of liver cirrhosis in the western world and is increasingly seen in Asian countries such as Japan and India.

Alcoholic hepatic steatosis, also called alcoholic fatty liver, consists in the occupation of a large proportion of the cytoplasm of affected hepatocytes by a single large triglyceride occlusion. This state is reversible if abstinence but may progress in cirrhosis if excess alcohol intake persist.

Alcoholic hepatitis is the second main step of alcoholic liver disease and associates steatosis together with inflammation and necrosis, due to excessive intake of alcohol.

Alcoholic cirrhosis is the most severe and terminal step of the alcoholic liver disease. It is characterized by fibrosis, leading to a progressive loss of liver function. Survival for patients affected by alcoholic cirrhosis is 60%-70% at one year and 35%-50% at five years.

There is no targeted therapy for this pathology, for which abstinence is known as the most important aspect of treatment. Corticosteroids are efficient in acute forms of alcoholic hepatitis but 40% of patients fail to respond to this treatment.

So, there is a real need for new strategies of treatment, with an early patient care for better results.

CB2 receptor has already been shown as blocking accumulation of human hepatic myofibroblasts (Julien B et al, 2005) and agonising the CB2 receptor has been proposed as a therapeutic strategy for the management of liver fibrosis (US20050143448).

On the other hand, CB2 receptors have been described as potentiating obesity-associated inflammation and complications such as insulin resistance and hepatic steatosis (Devaux V et al, 2009; WO2006138656).

It would be interesting to find new strategies of treatment for an early stage of alcoholic liver disease, knowing that the mechanisms are really distinct of those implicated in obesity-associated liver disorders.

SUMMARY

The present invention relates to a selective agonist of CB2 receptor for use in the prevention or treatment of alcoholic liver disease.

Particularly, the present invention relates to a selective agonist of CB2 receptor for use in the prevention or treatment of alcoholic hepatic steatosis and liver inflammation.

The present invention also relates to a pharmaceutical composition comprising a selective agonist of CB2 receptor for use in the prevention or treatment of alcoholic liver disease.

Particularly, the present invention relates to a pharmaceutical composition comprising a selective agonist of CB2 receptor for use the prevention or treatment of alcoholic hepatic steatosis and liver inflammation.

DETAILED DESCRIPTION

The inventors have shown that CB2 receptor activation triggers a decrease of the severity of alcoholic hepatic steatosis and the associated hepatic inflammation. Thus, the invention relates to the use of selective CB2 receptor agonists for the prevention or treatment of the early stage of alcoholic liver disease.

DEFINITIONS

The term “receptor agonist” has its general meaning in the art and refers to a natural or synthetic compound which binds the receptor to form a receptor-agonist complex activating said receptor and receptor-agonist complex, respectively, initiating a pathway signaling and further biological processes.

The term “CB2 receptor” or “cannabinoid receptor 2”, also named Cnr2 or CX5, has its general meaning in the art (Pertwee, R G, 1999) and refers to a G-protein coupled receptor encoded by the Cnr2 gene. The term may include naturally occurring “CB2 receptor” and variants and modified forms thereof. The CB2 receptor can be from any source, but typically a mammalian (e.g., human and non-human primate) CB2 receptor, particularly a human CB2 receptor. An exemplary native human CB2 receptor amino acid sequence is provided in GenPept database under accession number NP—001832 and an exemplary native human nucleotide sequence of Cnr2 mRNA is provided in GenBank database under accession number NM—001841.

The term “CB2 agonist” or “CB2 receptor agonist” refers to a compound able to activate CB2 receptor, i.e. able to specifically bind the CB2 receptor and trigger various signal transducing activities of the receptor.

The term “selective CB2 agonist” or “selective CB2 receptor agonist” refers to a compound able to selectively activate CB2 receptor. In the context of the present invention, CB2 agonists are selective for the CB2 receptor as compared with the CB1 receptor. By “selective” it is meant that the affinity of the agonist for the CB2 receptor is at least 10-fold, preferably 25-fold, more preferably 100-fold and still preferably 300-fold higher than the affinity for the CB1 receptor. The affinity of an agonist for CB2 (or CB1) receptor may be quantified by measuring the activity of CB2 (or CB1) receptor in the presence a range of concentrations of said agonist in order to establish a dose-response curve. Accordingly, a selective CB2 agonist is a compound for which the ratio Kd CB1/Kd CB2 is above 10:1, preferably 25:1, more preferably 100:1, still preferably 300:1.

The agonistic activity of compounds towards the CB2 (or CB1) receptors may be determined using various methods. For example, it is known that CB1/CB2 receptors are negatively G coupled protein receptors and CB1/CB2 agonists are thus capable of inhibiting the adenylate cyclase activity. Thus, the affinity of an agonist for CB2 (and CB1) receptor may be assayed by determining the ability of said agonist to block the effect of the Forskolin (an adenylate cyclase activator) in a cAMP measurement assay. In particular, a cAMP accumulation measurement assay has been described in Rinaldi-Carmona et al. (1998) in view of Matsuda et al. (1990) and Rinaldi-Carmona et al. (1996). Typically, CHO cells stably transformed with CB1 or CB2 are grown to confluence are washed with PBS and incubated for 15 min at 37° C. in 1 ml of PBS (containing 0.25% acid-free BSA, 0.1 mM IBMX, 0.2 mM RO20-1724) in the absence or in the presence a potential selective CB2 agonist to be assayed (for instance 10−9-10−6M). Forskolin (3 μM final concentration) is added and cells are incubated for another 20 min at 37° C. The reaction is terminated by rapid aspiration of the assay medium and addition of 1.5 ml of ice-cold 50 mM Tris-HCl, pH 8, 4 mM ethylenediaminetetraacetic acid. Dishes are placed on ice for 5 min and then the extracts are transferred to a glass tube. Extracts are boiled and centrifuged for 10 min at 3500 g to eliminate cell debris. Aliquots from supernatant are dried and the cAMP concentration is determined according to any suitable method and compared in the presence or absence of the selective CB2 agonist candidate. The one skilled in the art may in particular make use of one of the many commercial kits available for cAMP measurement.

In its broadest meaning, the term “treating” or “treatment” refers to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

In its broadest meaning, the term “preventing” or “prevention” refers to preventing the onset of a disorder in a subject. Particularly, the term “preventing an alcoholic liver disease” or “prevention of an alcoholic liver disease” refers to preventing the onset of a disorder in a subject susceptible to develop the disease, who is an alcoholic subject.

As used herein, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a subject according to the invention is a human.

According to the invention, the subject is a patient affected or susceptible to be affected by an alcohol liver disease, particularly at the step of alcoholic hepatic steatosis.

As used herein, the term “control” denotes a healthy subject and particularly a subject not affected by a hepatic disease, more particularly an alcoholic liver disease.

The term “biological sample” is used herein in its broadest sense. A biological sample is generally obtained from a subject. Frequently, a sample will be a “clinical sample”, i.e., a sample derived from a patient.

Therapeutic Methods and Uses

A first object of the invention relates to a selective CB2 agonist for use in the prevention or treatment of alcoholic liver disease.

In one embodiment, the invention relates to a selective CB2 agonist for use in the prevention or treatment of alcoholic hepatic steatosis and liver inflammation.

In one embodiment, the selective CB2 agonist of the invention may be a low molecular weight antagonist, e.g. a small organic molecule.

The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

Selective CB2 agonists are well known by the skilled man in the art.

A selective CB2 agonist that is contemplated by the invention is JWH-133 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6Hdibenzo[b,d]pyran] (see, e.g., Huffman et al, 1999). The structure of JWH-133 is provided here below:

Another selective CB2 agonist that is contemplated by the invention is JWH-015 [(2-Methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone] that is a drug from the aminoalkylindole family.

Another selective CB2 agonist that is contemplated by the invention is HU-308 [[(1R,2R,5R)-2-[2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl]-7,7-dimethyl-4-bicyclo[3.1.1]hept-3-enyl]methanol] is a selective CB2 agonist (L Hanus et al, 1999).

The compounds L-759,633 [(6aR,10aR)-1-methoxy-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromene] and L-759,656 [(6aR,10aR)-1-methoxy-6,6-dimethyl-9-methylidene-3-(2-methyloctan-2-yl)-7,8,10,10a-tetrahydro-6aH-benzo[c]chromene] are selective CB2 agonists that are contemplated by the invention.

The US patent application number US 2005165118 discloses pinene derivatives that can also be used. Such compounds have the following formula:

wherein: A---B designates an optional double bond, R1 is —CH2OH; G is hydrogen or —OR2 wherein R2 is a lower alkyl group; and R3 is a straight or branched chain C5-C12 alkyl.

Other selective CB2 receptor agonists that can be used include those disclosed in published US patent application US2004034090, entitled “3-Arylindole derivatives and their use as CB2 receptor agonists”. Agonists of this class have the following general structure:

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